14-gene assay interrogates circulating tumor cells

Liquid biopsies are opening new avenues for cancer research and diagnosis, but even experts in the field continue to nail down the best protocols for analyzing the cell-free DNA and circulating tumor cells (CTCs) contained in these peripheral blood samples.

Evi Lianidou, professor of Analytical Chemistry and Clinical Chemistry at the University of Athens, is one of those scientific pioneers. As early as 2005, she geared up to investigate the utility of gene expression analysis in CTCs. Her group, in collaboration with Clinical Oncologists in Greece, were the first to show the prognostic significance of CTC detection in early breast cancer. However, beyond detection and enumeration of CTCs, she was mostly interested in characterizing these rare cells at the molecular level. Using RT-qPCR, she evaluated the expression of a few cancer genes in these cells, but the number of important gene markers to be analyzed in these cells was continuously growing. But she quickly encountered a problem: the classic microarrays she expected to use for her project were not sensitive enough to be applied in such a low number of cells. Casting about for an alternative, she encountered xMAP® Technology from Luminex. She adopted the bead-based platform for multiplex gene expression analysis and has been deploying it for game-changing studies ever since. For instance, the scientists at the Analysis of Circulating Tumor Cells Lab applied xMAP Technology to simultaneously interrogate the expression of six genes in CTCs, as well as DNA methylation markers in these cells.

Wealth of Data

Moreover, CTCs are highly heterogeneous, so bulk population analysis could not achieve what Lianidou needed. Today, Lianidou and her team have developed a 14-gene assay to interrogate CTCs. Unlike existing FDA-cleared CTC assays which enumerate cells but fail to offer a molecular characterization, Lianidou’s xMAP-powered assay generates a wealth of data about gene activity even at the single-cell level. One of the reasons she built this assay on xMAP Technology is the ease with which assays can be refined or expanded later. “We want to have a lot more than 14 genes,” she says, noting that new information about cancer biology, cell therapy biomarkers, drug targets, and more can all be quickly added to the multiplex panel as it becomes available. Her team is currently evaluating the assay’s performance on clinical samples.

Those clinical samples are one of the reasons that Lianidou has been such a champion of xMAP Technology. With this type of research, she says, scientists don’t have the luxury of running more than one test on a sample because the input amounts are so small. With the Luminex platform, “we can test many targets in the sample at the same time,” Lianidou says. “It’s very important for high-throughput analysis to get a lot of information from these precious samples.”

Quick Tip

As a longtime developer of xMAP assays, Lianidou offers a simple tip for novice users: don’t skip the in silico step. “We always start from the in silico analysis of primers,” she says. Before her team even thinks about moving into chemistry, they carefully check to make sure all target sequences are unique in the genome and that primers will not interact with anything unexpected. “Specificity is a very important issue,” she says. “Make sure you ask, ‘Do we detect only this target and nothing else?’ And make sure no target is missing.”